1. Field of the Invention
This invention relates to a method and apparatus for detecting the presence of live microorganisms in gaseous streams. In one aspect, this invention relates to a portable apparatus for detecting the presence of live microorganisms in gaseous streams. In one aspect, this invention relates to a method and apparatus for detecting the presence of live microorganisms in gaseous streams produced by renewable energy sources, such as biogas and biomethane (a high methane content biogas) gas streams. In one aspect, this invention relates to a method and apparatus for detecting live microorganisms including, but not limited to, heterotrophic anaerobic bacteria, sulfate-reducing bacteria (SRB), acid-producing bacteria (APB), heterotrophic aerobic bacteria, and other types of culturable organisms having distinct metabolic characteristics as an indicator of positive growth.
2. Description of Related Art
As used herein, the term “biogas” refers to gases produced from renewable organic materials, also referred to as biomass, such as wood, agricultural crops or wastes, and municipal wastes. Biogas is produced from anaerobic degradation/digestion of organic matter, a process in which bacteria convert degradable organic matter into methane and carbon dioxide with smaller amounts of hydrogen sulfide, ammonia, and other trace components. Depending on the biomass source materials, produced biogas may contain constituents and compounds that pose hazards to human health, the environment, and safety. In addition, insufficiently processed biogas may contain trace or residual compounds that may compromise the integrity and operation of gas utilization equipment and/or natural gas pipeline systems. One such concern is the microorganisms which may be carried over from anaerobic digestion and which have survived the cleaning processes. When introduced into pipelines, some microorganisms may pose a corrosion risk to the pipelines. Thus, it is important that the overall microorganism load and major corrosion-causing microorganisms in biogas be identified, monitored, and removed prior to introduction of the biogas into existing natural gas supplies to avoid potential problems.
Conventional air sampling for the detection of microbes typically requires special equipment and highly trained personnel in specialized laboratories. In the U.S. pharmaceutical and medical industry, the most common air monitoring practice involves the use of impaction and centrifugal air samplers to collect a predetermined volume of air by passing the air sample through a microbial sampler and impacting the microorganisms in the air sample against agar-based microbial growth medium. Once the sample has been collected, the medium is incubated at a certain temperature for a predetermined period of time for detection and colony count of target microorganisms. For this purpose, there are a number of commercially available samplers designed for use in cleanroom environments. Slit-to-agar samplers, sieve impactors, sterilizable microbiological atrium, surface air system sampler, and MAS-100 microbial air monitoring systems are all commercially available samplers having similar design and sampling mechanisms. In each system or device, the sampler consists of a container designed to accommodate a Petri dish containing nutrient agar. A vacuum pump or fan draws a known volume of air through small holes or slits at the top of the sampler, and the particles in the air containing microorganisms impact on the agar medium in the Petri dish. The colonies are counted after the proper incubation period. The impaction speed as well as the particle size efficiency is a function of the diameter of the holes in the perforated lid and the speed of the fan. These samplers with high air flow rates generally allow for efficient impaction of particles 0.5 microns or larger in diameter, which is one of the major limitations for this type of technology, since many bacteria are smaller than 0.5 microns in diameter and, thus, cannot be efficiently captured by nutrient plates. In addition, the required high air flow, on the order of about one (1) cubic foot per minute, is not always possible to achieve in renewable gas lines, thereby further biasing the impaction toward the selection of larger particles. Finally, air flow desiccates the nutrient plate, limiting the maximum air sample volume that is allowed to pass each nutrient plate.
In centrifugal samplers, a propeller or turbine is used to pull a known volume of air into the unit and propel the air outward to impact on a tangentially disposed nutrient agar strip set on a flexible plastic base. However, the centrifugal air samplers disturb laminar airflow patterns and have been shown to have selectivity for larger particles as well, resulting in overestimation of the total airborne counts.
In liquid impingers, air is aspirated into a liquid and the microorganisms retained in the liquid are captured on a membrane filter through filtration and transferred to media plates to evaluate growth. The major shortcoming of the liquid impingement method is that the air bubbles in the headspace of the medium container burst and the majority of microorganisms in the air sample are not retained in the liquid medium. The retention rate of microorganisms is significantly affected by the volume of liquid medium (or the length of the path the bubbles travel in the liquid) and the size of bubbles generated during impingement. In addition, the multiple handling steps during sample collection may result in false positive results due to contamination.
In gelatin filtration, a gelatin filter is used to retain airborne microorganisms in the air pulled in by a vacuum pump. After a specific exposure time, the filter is asceptically removed and dissolved in an appropriate diluent and then plated on an appropriate agar medium to estimate its microbial content. Similar to nutrient plate sampling, the desiccation caused by the air flow limits the maximum air sample volume, and the larger pore size of the gelatin filter results in a bias towards larger particles and a lower recovery rate of microorganisms. In addition, as with the liquid impinger method, the multiple handling steps during sample collection may result in false positive results due to contamination.